Homodimer-mediated phosphorylation of C/EBPα-p42 S16 modulates acute myeloid leukaemia differentiation through liquid-liquid phase separation

CCAAT/enhancer binding protein α (C/EBPα) regulates myeloid differentiation, and its dysregulation contributes to acute myeloid leukaemia (AML) progress. Clarifying its functional implementation mechanism is of great significance for its further clinical application. Here, we show that C/EBPα regulates AML cell differentiation through liquid-liquid phase separation (LLPS), which can be disrupted by C/EBPα-p30. Considering that C/EBPα-p30 inhibits the functions of C/EBPα through the LZ region, a small peptide TAT-LZ that could instantaneously interfere with the homodimerization of C/EBPα-p42 was constructed, and dynamic inhibition of C/EBPα phase separation was observed, demonstrating the importance of C/EBPα-p42 homodimers for its LLPS. Mechanistically, homodimerization of C/EBPα-p42 mediated its phosphorylation at the novel phosphorylation site S16, which promoted LLPS and subsequent AML cell differentiation. Finally, decreasing the endogenous C/EBPα-p30/C/EBPα-p42 ratio rescued the phase separation of C/EBPα in AML cells, which provided a new insight for the treatment of the AML.

6.In Fig. 5C, we see S16 and C/EBPα-pS16 levels decrease over the 24 hr period of PMA treatment, while the ratio of pS16/S16 increases.The authors do not comment on why C/EBPα levels decrease at all.Is there degradation of the protein?The same is not seen in Fig. 5B, where C/EBPα-S16 levels remain constant while C/EBPα-pS16 increases upon Mannitol treatment (i.e., which induces condensate formation).This discrepancy should be addressed.7. Similarly, comparing the 24h timepoint in Fig. 5C to the 24h PMA treatment in Fig 5E shows completely different behavior of the S16 levels, which are essentially nonexistent in 5C at 24 hrs but still quite high in 5E.This discrepancy should be addressed.
8.An important observation made by the authors is that heterodimer formation of full-length C/EBPα with p30 reduces condensate formation, but they do not address the issue of the stoichiometry of the two protein forms.What mole ratio of p30 to full-length C/EBPα is needed to inhibit condensate formation?The authors should address this issue.9. Fig. 5G shows representative images comparing condensate formation associated with expression of WT C/EBPα vs. the S16E and S16A mutants.The images for WT vs. S16A appear to be reflective of the quantification shown.Again, this is why the method of puncta quantification used by the authors should be explained and validated.The same is true of the cell images shown in Fig. 6G.10.The qRT-PCR panel in Fig. 5H does not include any information on the cell type.This should be added to the legend or figure .11. Figure 6A-C does not include any information on the cell type.This should be added to the legend or figure.

REVIEWER COMMENTS
Reviewer #1 (Remarks to the Author): In the present manuscript, Wang et al demonstrate that CEBPA appear to undergo liquid-liquid phase separation (LLPS).Moreover, they claim that this is driven by formation of CEBPA-p42 homodimers, that the process is dependent on phosphorylation at S16 and that it can be inhibited by expression of CEBPA-p30.
While the demonstration of CEBPA-p42 undergoing LLPS is certainly novel the manuscript, as it stands, has a number of serious flaws and also lack demonstration of the functional relevance of their findings.

Response:
We thank the reviewer for the professional comments and thoughtful suggestions, which helped improve the manuscript.We have addressed these critiques and highlighted all the changes in yellow in the revised manuscript.Please find our point-by-point responses below.

Major concerns
1.The authors seem to ignore a significant amount of recent literature on the function of CEBPA yielding a non-contemporary presentation of the field.Statements such as "….mechanism by which CEBPA functions is still unclear…" and that CEBPA-p30 should be "dominant negative" and that "CEBPA-p30 acts mainly by inhibiting the formation of CEBPA homodimers" are simply wrong.
Response: Thank you very much for your valuable and constructive comments, we apologize for the inaccurate description.We have reviewed the recent literatures on the function of C/EBPα and found that C/EBPα enhanced cell differentiation and blocked the cell cycle progression by blocking the miR-182 expression, activating CSF3R or IL6R, repressing CEBPG and E2F, respectively [1][2][3][4][5][6][7] .More importantly, C/EBPα was necessary for long-term self-renewal and lineage initiation of hematopoietic stem cells 8 , and improved the curative effects of the LSD1 inhibitor in the treatment of AML 9 .
In addition, its mutation has been widely used in the clinical diagnosis and treatment of AML 10,11 .In summary, the functions of C/EBPα in various pathophysiological processes have been clarified.Further clarification of the specific molecular mechanism to achieve its functions is of great significance for the development of related drugs targeted at C/EBPα.We have revised the related descriptions in the new manuscript.
(Line 18-19, 35-42) Meanwhile, we also confirmed that, different from the "dominant negative" effect of C/EBPα-p30 in normal genotype cells which expressed C/EBPα-p42 and C/EBPα-p30 simultaneously, it works independently of C/EBPα-p42 in most patients with C/EBPα mutation.For example, C/EBPα-p30 sustained leukemic growth via the CD73/A2AR axis 12 ; it alleviated immunosuppression of CD8 + T cells by inhibiting autophagyassociated secretion of IL-1β in AML 13 .Thanks for your suggestion and we have modified the descriptions in this revised manuscript (Line 45-59).2. Also the authors should use CEBPA-p42 homodimers (instead of CEBPA homodimers) as CEBPA-p30 can dimerize with CEBPA-p42 which could, in principle"be termed CEBPA homodimers.
Response: Thanks for your suggestion.We have revised the inaccurate description of C/EBPα homodimers to C/EBPα-p42 homodimers throughout the article.
3. The authors seem to think that role of CEBPA-p30 in CEBPA mutant AML is to prevent the formation of CEBPA-p42 homodimers.This is not correct as the CEBPA mutant AML is bi-allelic mutated and are either homozygous for the CEBPA-p30 allele or carries on CEBPA-p30 allele and an allele blocking CEBPA (any) dimerization.In essence this result in a situation where CEBPA-p30 homodimers are the only functional CEBPA dimers in these patients.The authors should modify the manuscript accordingly.
Response: Thank you for pointing out this issue.As you and some reports 1-3 mentioned, the majority of AML patients with CEBPA mutation are biallelic mutations.The selective loss of C/EBPα-p42 was found in these patients because they usually have one allele carrying the N-terminal mutation and the other carrying the C-terminal mutation.As we all know, the N-terminal mutations often expressed C/EBPα-p30 which could form C/EBPα-p30 homodimers, and the C-terminal mutations could either block dimerization of C/EBPα with itself or with other members of the CEBP family.Thus, C/EBPα-p30 homodimers are the only functional C/EBPα dimers in these patients.We are sorry for our inaccurate description of AML patients with C/EBPα mutation, and we have revised the relevant descriptions in the new manuscript according to your suggestions (Line 45-59).Response: Thank you for the suggestion.Actually, most reports [1][2][3][4][5] proved LLPS by using different materials, such as purified protein, overexpressed protein in pattern cells and endogenous protein under physiological conditions.In this study, we tried to add mannitol to induce the LLPS of overexpressed-C/EBPα-EGFP in HEK 293T.Then, we also found endogenous C/EBPα undergoes LLPS in PMA-treated THP-1 cells, various hormones-treated 3T3-L1 cells and G-CSF-treated 32Dcl3 cells.The three induceddifferentiation cell models fully confirmed the behavioral performance of C/EBPα during the process of cell differentiation.Furthermore, endogenous C/EBPα condenses were observed in AML cells.And our peptide, which had been proved to inhibit LLPS of C/EBPα in HEK293T cell and induced-differentiation cell models, was also found to inhibit the formation of endogenous C/EBPα condenses without any stressors in vitro and in vivo (Fig. 4g, 6b and h).In summary, we believed that C/EBPα is also normally undergoing LLPS in steady state.The authors should also make an effort to define why LLPS is measured in the way they do as this is not common knowledge in the field.

References:
Response: Thank you for pointing out the issue.Many methods have been established to quantitatively describe the LLPS [1][2][3][4][5][6] .Quantifying the number of particles in each cell is commonly used to study the difference of protein phase separation.Therefore, we counted the number of particles in each cell to characterize the phase separation.
Relative descriptions have been added in the new Methods section (Line 579-591).Response: We appreciate your thorough comments.In order to confirm the influence of phase separation on transcription function of C/EBPα, we performed ChIP-seq and RNA-seq for PMA treated THP-1 cells.The results of ChIP-seq showed that the binding ability of phase-separated-C/EBPα with many reported downstream target genes CSF3R 1-2 , IL6R 3 and CEBPG 4 was significantly enhanced (Supplementary Fig. 2f).

References
Meanwhile, the results of RNA-seq showed that phase-separated-C/EBPα activated the transcription of CSF3R and IL6R, but inhibited the transcription of CEBPG (Supplementary Fig. 2g).All the results were consistent with the previously reports and proved that the transcriptional role of C/EBPα can be activated by its LLPS.The results and relative descriptions have been added in the new version (new Supplementary Fig.    6.As CEBPA-p30 can also dimerize with itself the authors should test if these dimers are undergoing LLPS Response: We thank you very much for your valuable and constructive comments.As your mentioned, the C/EBPα-p30 homodimers was observed (Fig. 4a), but no condensate was found in mannitol stimulated-HEK 293T cells that were transfected with mCherry-tagged C/EBPα-p30.The results suggested that C/EBPα-p30 homodimers cannot undergo LLPS.We have added the results and relative descriptions to the revised manuscript (new Supplementary Fig. 4b, line 188-190).Response: Thank you for pointing out this issue.According to your suggestion, we detected the endogenous LLPS of C/EBPα in two normal differentiation systems [1][2][3] .
Firstly, when the 3T3-L1 cells differentiated into adipocytes, the endogenous C/EBPα condensates were significantly increased (Supplementary Fig. 1d-e).Then, during the differentiation of 32Dcl3 cells into granulocytes, the increase of endogenous C/EBPα condensates were also observed (Supplementary Fig. 1f-g).These results proved that endogenous C/EBPα undergoes LLPS in normal differentiation systems.We have added these results to the revised manuscript (new Supplementary Fig. 1d-g; Line 96-101).

References:
1. Bo TP, Pedersen TA, Xu X, Bo L, Nerlov C. E2F repression by C/EBPalpha is required for adipogenesis and granulopoiesis in vivo.Cell 107, 247-258 (2001).8.The authors findings in the MLL-AF9 mouse model are puzzling as it has been shown that CEBPA can be deleted in these cells without any apparent functional consequences.
These findings suggest that the data presented in Figure 6 is not related to CEBPA function but perhaps to other CEBPs.
Response: Thank you very much for your valuable and constructive comments.Porse and Tenen had used C/EBPα wild-type and knock-out MLL/AF9 mouse model to confirm that C/EBPα is required for AML initiation, but not for AML maintenance [1][2][3] , which proved that C/EBPα expressed normally in MLL/AF9 mouse and played important role in AML differentiation.In our study, Ara-C and DOX were employed to induce the differentiation of MLL-AF9 AML cells in the present of endogenous C/EBPα (Supplementary Fig. 6c).The restored LLPS of C/EBPα was observed, which further proved the functions of C/EBPα in AML differentiation (Supplementary Fig. 6c; Line 304-306).Meanwhile, in order to prove our conclusion, we established AML xenograft mouse models by injecting NSG mice with mScarlet tagged LZoverexpressed THP-1 cells.Expectedly, the results were identical with that in MLL-AF9 AML mouse model (Supplementary Fig. 6e-j).In summary, we believe that the LLPS of C/EBPα-p42 homodimers promotes AML differentiation.We have added the results and relative descriptions in the new version manuscript (new Supplementary Fig. 6e-j; Line 313-316). References:

Response:
We appreciate the reviewer's comments.As your suggestion, we detected the phase separation of other C/EBPs proteins and found that C/EBPδ, C/EBPε and C/EBPγ could not undergo LLPS after being 10 min hyperosmotic stress stimulation (Supplementary Fig. 2e).We have added these results to the revised manuscript (Supplementary Fig. 2e; Line 141-146).9.The authors claim that the effect of rapamycin is via downregulation of CEBPA-p30.
However, rapamycin has MANY effects and should therefore modify their conclusion to account for other possibilities.
Response: Thank you for pointing out the issue.We agreed with the reviewer that rapamycin regulates AML through a variety of pathways, such as inhibiting AML cell viability via circ_0094100/miR-217/ATP1B1 axis 1 or enhancing the anti-leukemia effect as a specific inhibitor of mTOR 2 .Therefore, we overexpressed C/EBPα-p30 in THP-1 cells treated with PMA and rapamycin.The results of qRT-PCR and flow cytometry analysis showed that C/EBPα-p30 reversed the differentiation promoting effect of rapamycin (Fig. 6c, d and Supplementary Fig. 6b), which suggesting that rapamycin promoted differentiation mainly by down-regulation of C/EBPα-p30.We have added these results and descriptions to the revised manuscript (Fig. 6c, d and Supplementary Fig. 6b; Line 292-299).

References:
1. Cao J, Huang S, Li X. Rapamycin inhibits the progression of human acute myeloid leukemia by regulating the circ_0094100/miR-217/ATP1B1 axis.Experimental hematology 112-113:60-69.e62(2022).Response: We thank the reviewer for raising this point.In the revised manuscript, we evaluated the relative levels of C/EBPα-p30 and C/EBPα-p42 in MLL-AF9 mice treated with rapamycin in combination with Ara-C and Dox or not.The results showed that rapamycin decreased the relative levels of C/EBPα-p30 to C/EBPα-p42.We have updated the figures and results in the revised manuscript (new Supplementary Fig. 6c, Line 304-306).
Minor points: 1. How many AML patients were tested in Fig1 D Response: Thanks a lot for your careful reading about our manuscript.We analyzed 6 AML patients, and at least 10 random fields were observed in each AML patient in Fig. 1d.We have added the descriptions to the revised manuscript (Line 92 and Line 846-848).

Fig 4G. It seems unlikely that the data presented there reach statistical significance
Response: We appreciate your detailed advice and we are sorry for the misunderstanding caused by our failure to describe the comparison.Because of the limited degree of difference, we re-synthesized the relevant primers, added the PMA untreated group as a control and restart the qRT-PCR experiment.The results still showing significance.We provided our original data and statistics as follows for your review, so as to increase the persuasive power of our data.Meanwhile, we have updated the figures and results at the corresponding location in the revised manuscript (new Response: We appreciate the reviewer's comments and apologize for the lack of some labels.In Fig. 6a, we used THP-1 cells to confirm that rapamycin regulates the ratio of C/EBPα-p30 to C/EBPα-p42 by decreasing the endogenous C/EBPα-p30 protein level. We have added it to the revised manuscript (Line 283 and Line 943).The key mechanism of the study is relatively complete, among which the effect of homologous dimer on the phosphorylation of S16 and LLPS of protein is the innovation point.The design of interference peptide and C/EBPα-p30/C/EBPα ratio, which is associated with the treatment of acute leukemia, has certain practical significance.
However, there was no direct experiment to prove the homodimers in this paper, and the function of the protein, especially its influence on the differentiation of AML cells, was not fully demonstrated.As for the key mutations in the research, such as C/EBPα-p30 and S16A/E, no relevant animal models were established for verification, which made the paper not persuasive enough to meet the publication requirements.

Response:
We thank the reviewer for the professional comments and thoughtful suggestions to help improve the manuscript.We have addressed these critics and highlighted all the changes as yellow in the revised manuscript.Minor comments 1.In line 94, "condensate" is not suitable for droplets in vitro, which is only used to describe LLPS in vivo.

Response:
We appreciate the reviewer's comments.According to your suggestion and the previous study 1 , we have changed the "condensate" to "micrometre-sized spheres" at the corresponding location in the revised manuscript (Line 114).

References:
1 The authors also identify a posttranslational modification of phospho-Serine 16 (pS16) in C/EBPα under conditions in which the protein forms robust condensates and they establish that higher pS16 levels correlate with enhanced condensation.Introducing a phospho-mimetic mutation at this site increases the number of condensates, while a phospho-null mutation decreases the number of condensates.To further support their model of C/EBPα-p30 leading to dysfunction through heterodimerization with fulllength C/EBPα, the authors utilize Rapamycin, which causes degradation of the C/EBPα-p30 protein, thus tuning the ratio of C/EBPα-p30 to C/EBPα.They find that this rescues full-length C/EBPα LLPS, which also rescues expression of differentiation markers.Overall, the data in this article is of high quality and the findings are convincing, although there are several concerns that should be addressed prior to publication.The content of the article will be of interest to scientists in the transcription and biomolecular condensates fields and is the first report linking LLPS with transcriptional and developmental regulation by C/EBPα.

Response:
We thank the reviewer for the professional comments and thoughtful suggestions, which helped improve the manuscript.We have addressed these critiques and highlighted all the changes in yellow in the revised manuscript.Please find our point-by-point responses below.
Major Points: 1.The authors repeatedly use mannitol-induced osmotic stress to induce condensation of C/EBPα in HEK293T cells, but never explicitly state why this treatment is used.This needs to be addressed in the text and citations showing that stress may be needed for transcription factor LLPS should be included.2. While the authors quantify the condensates formed by C/EBPα throughout the paper, the Methods section only states that this was done using FIJI.The authors should elaborate on the quantification method (i.e., thresholding, image processing, etc.).
Additionally, the authors should show the quality of their puncta segmentation in the form of side-by-side images of the raw GFP-C/EBPα channel and the segmented puncta after quantification.These data should be shown in supplemental figures for all sets of images.This is to ensure that the condensates are being accurately counted.

Response:
We thank you very much for your valuable and constructive comments.
Based on your suggestion, we have updated the methods (Line 577-589) and showed the raw GFP-C/EBPα channel and the segmented puncta after quantification in the supplemental figures.Specific as follows: A set of fluorescence images from the experiment were obtained using the same microscope settings (such as resolution, optical zoom, gain, laser intensity and scan speed) to ensure consistency between the sample and the experiment.The original picture was imported into the Image J and converted into 8bit, and then adjusted the parameters to accurately identify particles.In order to accurately count the number of particles, we set different thresholds according to different experimental materials.For example, the threshold is set to 50-255 gray level for phase-separated particles produced by overexpressed-C/EBPα in HEK 293T and THP-1 cells.For endogenous C/EBPα particles, set the thresholds of MLL-AF9 cells, AML primary cells, 3T3-L1 cells and 32Dcl3 as 90-255, 75-255, 85-255 and 60-255, which effectively eliminated the background signals in the analysis.The numbers of puncta of each cell within these parameters were evaluated.We have added the descriptions to the Method section of the revised manuscript (Line 579-591).
3. The authors go to great lengths to show that LLPS of C/EBPα is at play during the differentiation induced by anti-leukemic drugs, but they never discuss an explicit mechanism.From their data, it is suggestive that C/EBPα forms active transcriptional condensates at genes that promote differentiation.The authors should discuss this in the Discussion section to highlight the likely mechanism underlying their findings.
Response: Thanks a lot for your careful reading on our manuscript.To comprehensively describe the regulation mechanisms of C/EBPα LLPS on cell differentiation, we performed ChIP-seq (ChIP-sequencing) and RNA-seq in PMAtreated THP-1 cells, in which C/EBPα undergoes LLPS.The ChIP-seq data showed that the binding ability of phase-separated-C/EBPα with downstream target genes CSF3R, IL6R and CEBPG was significantly enhanced (Supplementary Fig. 2f).Simultaneously, RNA-seq results showed that C/EBPα activated the transcription of CSF3R and IL6R, but inhibited the transcription of CEBPG (Supplementary Fig. 2g).These targets have been reported as the direct downstream molecules of C/EBPα [1][2][3][4] , and the results proved

Response:
We appreciate the reviewer's comments and apologize for our rough description.Actually, in Fig. 5C, we used THP1 to detect endogenous C/EBPα p-S16 levels.However, in Fig. 5E, we used plasmids mediated C/EBPα overexpression system to detect the effect of C/EBPα-p30 or LZ on phosphorylation of C/EBPα.We have provided a more detailed description at the corresponding location in the revised manuscript (Line 237-245).
8.An important observation made by the authors is that heterodimer formation of fulllength C/EBPα with p30 reduces condensate formation, but they do not address the issue of the stoichiometry of the two protein forms.What mole ratio of p30 to fulllength C/EBPα is needed to inhibit condensate formation?The authors should address this issue.
Response: Thanks a lot for your careful reading about our manuscript.After calculation, the mole ratio of C/EBPα-p30 to full-length C/EBPα is 12.6: 1, which could inhibit the condensate formation.We have added it to the figure legends in the revised manuscript (line 882).9. Fig. 5G shows representative images comparing condensate formation associated with expression of WT C/EBPα vs. the S16E and S16A mutants.The images for WT vs. S16A appear to be reflective of the quantification shown.Again, this is why the method of puncta quantification used by the authors should be explained and validated.
The same is true of the cell images shown in Fig. 6G.

Response:
We thank the reviewer for the suggestions.Based on your suggestion, we have added the puncta quantification in the revised manuscript (new supplementary Fig. 4d and 5d).Specific as follows: A set of fluorescence images from the experiment were obtained using the same microscope settings (such as resolution, optical zoom, gain, laser intensity and scan speed) to ensure consistency between the sample and the experiment.The original picture was imported into the Image J and converted into 8bit, and then adjusted the parameters to accurately identify particles.In order to accurately count the number of particles, we set different thresholds according to different experimental materials.For example, the threshold is set to 50-255 gray level for phaseseparated particles produced by overexpressed-C/EBPα in HEK 293T and THP-1 cells.
For endogenous C/EBPα particles, set the thresholds of MLL-AF9 cells, AML primary cells, 3T3-L1 cells and 32Dcl3 as 90-255, 75-255, 85-255 and 60-255, which effectively eliminated the background signals in the analysis.The numbers of puncta of each cell within these parameters were evaluated.We have added the descriptions to the Method section of the revised manuscript (Line 579-591).
10.The qRT-PCR panel in Fig. 5H does not include any information on the cell type.
This should be added to the legend or figure.
Response: We thank the reviewer for pointing this out.In Fig. 5H, we used the THP-1 cells that overexpressing C/EBPα-S16E-EGFP, C/EBPα-EGFP (WT) or C/EBPα-S16A-EGFP to study their influences on the cell differentiation.We have added the description to the legend in the revised manuscript (new Fig. 5f; line 929-931).
11. Figure 6A-C does not include any information on the cell type.This should be added to the legend or figure.
Response: We appreciate the reviewer's comments and apologize for the lack of some labels.In Fig. 6a, we used THP-1 cells to confirm that rapamycin regulates the ratio of C/EBPα-p30 to C/EBPα by decreasing the endogenous C/EBPα-p30 protein level.In Fig. 6b & 6c, we also used THP-1 cells with stable expression of mScarlet tagged LZ.
We have thoroughly examined similar issues and added the description of cell type to the corresponding legend in the revised manuscript (new Fig. 6a-c, Line 283,942-951).

3 .
Jin X, Zhou M, Chen S, Li D, Cao X. Effects of pH alterations on stress-and aginginduced protein phase separation.Cellular and Molecular Life Sciences 79, 380 (2022).

4 .
Matos C, et al.Liquid-liquid phase separation and fibrillation of the prion protein modulated by a high-affinity DNA aptamer.FASEB journal: official publication of the Federation of American Societies for Experimental Biology 34, 365-385 (2020).5. Guo C, et al.ENL initiates multivalent phase separation of the super elongation complex (SEC) in controlling rapid transcriptional activation.Science advances 6, eaay4858 (2020).6. Gao H, et al.Phase separation of DDX21 promotes colorectal cancer metastasis via MCM5-dependent EMT pathway.Oncogene, (2023).

5 .
The authors seem to completely forget the transcriptional role of CEBPA.Does this change if they add mannitol to cells as determined by RNA-seq and CHIPseq?

3 .
Tenen DG, Darlington GJ, Link DC, Zhang P, Iwama A, Datta MW.Upregulation of interleukin 6 and granulocyte colony-stimulating factor receptors by transcription factor CCAAT enhancer binding protein alpha (C/EBP alpha) is critical for granulopoiesis.The Journal of Experimental Medicine, 188 (1998).

7 .
The authors should test if endogenous CEBPA is undergoing LLPS in a normal differentiation system.The authors should also test the impact of the S16 mutation in differentiation (NIH3T3-mediate adipocyte; see Porse et al, 2001 or 32Dcl1) systems driven by exogenous addition of CEBPA.

3 .
Strom D, Cleveland J, Chellappan S, Nip J, Hiebert S. E2F-1 and E2F-3 are functionally distinct in their ability to promote myeloid cell cycle progression and block granulocyte differentiation.Cell Growth & Differentiation: the Molecular Biology Journal of the American Association for Cancer Research 9, 59-69 (1998).

3 .
Avellino R, Delwel R. Expression and regulation of C/EBPα in normal myelopoiesis and in malignant transformation.Blood 129, 2083-2091 (2017).This begs the question of whether other CEBPAs can undergo LLPS.In fact, the LZ peptide would predict to influence other CEBPs as well as CEBPs are well-known to heterodimerize with each other.

2 .
Liesveld JL, Rosell K, Lu C, Mulford D, Walker A. The mTOR Inhibitor Rapamycin Demonstrates Activity Against AML in Combination with Imatinib Mesylate and with 5-Azacytidine.Blood 110, 4318-4318 (2007).Also, to support the data in the MLL-AF9 mouse models (Fig 6) the authors should perform westerns to assess the relative levels of CEBPA-p30 and CEBP-p42.
is a transcription factor associated with myeloid differentiation, and functional abnormalities such as C/EBPα-p30 mutation can lead to acute myeloid leukemia (AML).Here the article shows that homodimers of C/EBPα regulate AML cell differentiation by influencing the phase separation by promoting phosphorylation at S16.However, the p30 mutation inhibits the LLPS of C/EBPα by inhibiting homodimer binding and S16 phosphorylation levels in WT via the LZ domain.According to this property, TAT-LZ, a membrane-penetrating peptide, was designed in this research to regulate cell differentiation by inhibiting the homodimer binding of proteins and the level of phase separation.

1 . 2 .
Below please find out point-by-point responses:Major comments Although it has been proved that C/EBPα-p30 mutation inhibits protein transcriptional activity by interfering with homodimerization which is a key link in this study, relevant experiments are still needed.For example, the TAT-LZ peptide affects the homodimer binding of C/EBPα, thus the binding force and expression level of the dimer can be characterized by NATIVE PAGE or MST methods, instead of assuming that proteins not bound to the peptide exist in the form of dimer.Response:We appreciate your thorough comments.Based on your suggestion, we performed experiment to confirm C/EBPα-p30 or LZ combined with C/EBPα to form heterodimers and inhibit the formation of C/EBPα homodimers.In detail, the HEK 293T cells were transfected with mCherry-tagged C/EBPα-p30 or LZ fragment together with C/EBPα constructs (Supplementary Fig.4a).By using NATIVE PAGE, C/EBPα-p42 monomer, C/EBPα-p30 monomer, C/EBPα-p42 homodimer, C/EBPα-p30 homodimer and C/EBPα-p42: C/EBPα-p30 heterodimer were observed (Fig.4a).More importantly, the interfering effects of C/EBPα-p30 or LZ for the formation of C/EBPα-p42 homodimer were proved.We have added the results and relative descriptions to the revised manuscript (Fig.4a, Line 185-188).As for the verification of protein function, only the transcription levels of CD11b and CD68 were used for characterization, which was not comprehensive and systematic.This study focuses on the regulation of C/EBPα on AML cell differentiation, so relevant functional experiments, especially flow cytometry experiments, are necessary, otherwise they are not convincing.Response: Thank you for pointing out this issue.We have added the flow cytometry experiments to analyze the ratio of CD11b or CD68 positive cells.The results supported our conclusion from qRT-PCR and had been added to the revised manuscript (Fig. 4j,5g and 6d).

3 .
In this study, there were several key disease-related gene mutations, such as p30 and S16A/E, and cell transfection by constructing plasmids alone was not sufficient to simulate the disease.The corresponding transgenic model of mice is a crucial part of this project.Response: Thank you for your valuable and constructive comments.According to your suggestion, we established the MLL-AF9 AML mouse models with Control, C/EBPα (WT), C/EBPα-p30, C/EBPα-S16E and C/EBPα-S16A groups.Compared with the Control group, we found that C/EBPα-p30 significantly promoted the progression of AML, while both C/EBPα S16E and C/EBPα (WT) inhibited the progression of AML and the inhibitory effect of C/EBPα S16E was stronger than that of the WT group.All the results proved the function of C/EBPα S16 phosphorylation-mediated LLPS on AML progression and improved our models.We have added the results to the revised manuscript (Fig. 5h-i, Line 268-271).
. Cai D, et al.Phase separation of YAP reorganizes genome topology for long-term YAP target gene expression.Nat Cell Biol 21, 1578-1589 (2019).Reviewer #3 (Remarks to the Author): The article by Wang, et al., proposes that liquid-liquid phase separation (LLPS) by the C/EBPα transcription factor in the nucleus is essential for promoting myeloid differentiation.The authors show through transient overexpression in HEK293T cells and immunostaining of endogenous C/EBPα in AML patient cells and THP-1 cells that upon drug-induced differentiation, C/EBPα localizes within punctate condensates.They show both in vitro and in HEK293T cells that these condensates exhibit properties associated with LLPS (turbidity, fusion events, rapid FRAP kinetics).An AMLassociated mutant of C/EBPα which lacks the N-terminal 120 residues (termed p30) is unable to undergo LLPS on its own and inhibits the LLPS of full-length C/EBPα.The authors show that this is due to heterodimerization of the truncated p30 with full-length C/EBPα via their leucine zipper domains, thus interfering with homodimerization of the full-length protein.Further, deletion of the leucine zipper domain within the truncated p30 (C/EBPα-p30 ΔLZ construct) rescues C/EBPα LLPS, while a peptide consisting of just the LZ domain phenocopies C/EBPα-p30 and inhibits C/EBPα LLPS.
that the LLPS of C/EBPα promoting the differentiation of AML cells by activating the transcriptional activity of its classical downstream molecules.Based on your suggestion, the added results and relative discussion have been added to the Results and Discussion sections of the revised manuscript.(Supplementary Fig. 2f-g, Line 148-154 and 365-368).7. Similarly, comparing the 24h timepoint in Fig. 5C to the 24h PMA treatment in Fig 5E shows completely different behavior of the S16 levels, which are essentially nonexistent in 5C at 24 hrs but still quite high in 5E.This discrepancy should be addressed.
1. Peggy, et al.Modeling of C/EBPα Mutant Acute Myeloid Leukemia Reveals a Common Expression Signature of Committed Myeloid Leukemia-Initiating Cells.
We thank you very much for your valuable comments.It has been reported that LLPS is a new mechanism by which transcription factors regulate transcription processes.Some proteins, such as YAP1 and SEUSS, should be activated by LLPS to